A two-step process for cytokine production revealed by IL-4 dual-reporter mice - PubMed (original) (raw)

A two-step process for cytokine production revealed by IL-4 dual-reporter mice

Katja Mohrs et al. Immunity. 2005 Oct.

Abstract

To monitor IL-4 expression at the single-cell level, we generated mice with insertions of different reporter genes into both copies of the Il4 gene that permitted the simultaneous analysis of IL-4 transcripts via GFP and IL-4 protein secretion by use of huCD2. Innate and adaptive cells competent for IL-4 production were marked by GFP, while cells that presently or recently secreted IL-4 additionally displayed huCD2. After challenge with the strictly enteric helminth, Heligmosomoides polygyrus, GFP-positive innate and adaptive cells disseminated widely, but IL-4 secretion was predominantly mediated by CD4+ T cells in the intestines and draining lymphoid organs. IL-4-competent cells persisted in cured animals, and memory responses reflected rapid cytokine production at the site of rechallenge. These data reveal a two-step process for cytokine production: the first generating poised cells that disseminate systemically and the second inducing the rapid production of the cytokine in response to local stimulation.

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Figures

Figure 1

Figure 1. Schematic of 4get/KN2 IL-4 dual-reporter mice

In heterozygous 4get/KN2 IL-4 dual-reporter mice one Il4 allele is marked by the addition of the bicistronic IRES-GFP reporter (4get). The first two exons of the other allele are replaced with the huCD2 reporter (KN2) cassette. 4get/KN2 mice have a functional copy of IL-4 as part of the bicistronic 4get allele. Filled boxes with numbers indicate exons. See Supplementary Fig. 1 for details.

Figure 2

Figure 2. huCD2 expression faithfully reflects IL-4 protein secretion

CD4+ T cells were purified from naïve 4get/KN2 mice and stimulated in the presence of APC under Th2 (IL-4 + anti-IFN-γ) or Th1 (IL-12 + anti-IL-4) polarizing conditions. (a) After 6 days these cultures were transferred to uncoated (resting) or anti-CD3ε-coated (αCD3) wells and analyzed the next day for huCD2 expression (top panel) or IL-4 secretion (bottom panel) using a cytokine secretion assay. (b) The plate-bound anti-CD3ε stimulation of Th2 polarized cultures was abruptly terminated by transfer to fresh, uncoated wells (0 h) and the cultures were analyzed 24 and 48 h later. Vertical numbers indicate the MFI of GFP+/huCD2+ population. (c) Th2 cultures were generated and restimulated as in (a) for the indicated periods of time (right panel). huCD2 expression and IL-4 secretion were determined as in (a). Representative FACS plot depicts the 8 h time point. Q1: IL-4-/huCD2+; Q2: IL-4+/huCD2+; Q3: IL-4+/huCD2-. All huCD2+ and/or IL-4+ cells were also GFP+ (see a, b and data not shown).

Figure 3

Figure 3. huCD2 expression reflects IL-4 protein secretion by lymphoid and nonlymphoid cells in vivo

(a) Naïve 4get/KN2 mice or 4get/+ littermate controls were injected i.v. with either PBS (0 min) or anti-CD3ε. Splenic NK T cells (NK1.1+/CD4+) or conventional (NK1.1-/CD4+) CD4+ T cells were analyzed 45 and 90 min later for the expression of GFP and huCD2. (b) Naïve 4get/KN2 mice were either injected with PBS or were sensitized by i.p. injection of anti-DNP mouse IgE. 24 h later the sensitized animals were injected i.p. with either PBS or DNP30-40-HSA. Basophils (FcεRI+/CD4-/SSClo) in the livers of all groups were analyzed 2 h later for the expression of GFP and huCD2.

Figure 4

Figure 4. GFP and huCD2 expression in response to infection with H. polygyrus

4get/KN2 mice were infected with H. polygyrus and analyzed 2 weeks later. (a) CD4+ T cells in the indicated organs were analyzed directly ex vivo for the expression of GFP and huCD2. (b) _Hp_-infected animals were injected i.p. with PBS (PBS), anti-CD3ε (αCD3), anti-IgE (αIgE), IL-3 + IL-18 or worm extract and sacrificed 2-4 h later. CD4+ T cells, basophils (FcεRI+/CD4-/SSClo), and eosinophils (CCR3+/CD4-/SSChi) in the liver were analyzed for the expression of GFP and huCD2. (c) Peritoneal exudate cells were cultured in the absence (no extract) or presence of crude extracts prepared from adult worm (worm extract), larvae extract or S. mansoni egg antigen (SmEA) (top panel). The next day CD4+ T cells were analyzed for the expression of GFP and huCD2. The same procedure was performed after mice were immunized 1 week earlier i.p. with S. mansoni eggs (bottom panel).

Figure 5

Figure 5. Phenotype of huCD2+ and huCD2- GFP+ Th2 cells

4get/KN2 mice were infected with H. polygyrus and the mesLN were analyzed 2 weeks later. CD4+ T cells with a GFP-/huCD2-, GFP+/huCD2-, or GFP+/huCD2+ phenotype (as indicated on the small dot plot to the left) were analyzed for the expression of the indicated surface markers (depicted in alphanumerical order).

Figure 6

Figure 6. Cytokine transcripts and protein production by huCD2+ and huCD2- GFP+ Th2 cells

4get/KN2 mice were infected with H. polygyrus and the mesLN were analyzed 2 weeks later. (a) CD4+ T cells with a GFP-/huCD2-, GFP+/huCD2-, or GFP+/huCD2+ phenotype were sorted from the mesLN. (b) mRNA was immediately prepared from the respective populations, reversed transcribed and analyzed by real-time RT-PCR for the indicated transcripts normalized to GAPDH. Depicted is the expression relative to GFP- cells. Shown are the mean± SD from triplicate samples. (c) The sorted populations (a) were cultured in the absence (0 h) or presence of plate-bound anti-CD3ε. Supernatants were harvested after 4 and 24 h and analyzed by ELISA for the indicated cytokines. The detection limits are indicated by the dotted line. Depicted are the mean± SD. The stimulated CD4+ T cells (c) were analyzed after 4 and 24 h for the expression of huCD2 (d) and GFP versus huCD2 after 24 h (e). (f) The sorted populations (a) were either directly stimulated for 4 h with PMA + ionomycin (P+I, left panel) or rested for 18 h prior to 24 h stimulation on plate-bound anti-CD3 (rested, right panel) and the supernatants were analyzed by ELISA for IL-4.

Figure 7

Figure 7. huCD2 expression is highly regulated during the course of primary and challenge Hp infection

(a) 4get/KN2 mice were infected with H. polygyrus (1° Hp) and CD4+ T cells in the mesLN were analyzed at the indicated time points for GFP (upper panels) and huCD2 (lower panels) expression. One cohort was drug-cured (Rx) after 2 weeks while the control group remained untreated. Animals from both groups were analyzed 5 weeks after drug treatment (7 weeks after 1° Hp). The remaining drug-cured animals were rechallenged (2° Hp) 5 weeks after drug treatment and analyzed at the indicated time points. Depicted are the mean± SD. (b) Naïve (primary) or drug-cured (recall) 4get/KN2 mice were infected with H. polygyrus and CD4+ Th2 cells (CD4+/FcεRI-/GFP+), basophils (FcεRI+/CD4-/GFP+), and eosinophils (CCR3+/GFP+) were analyzed at various time points in the indicated organs for huCD2 expression. (c) Naïve (primary) or drug-cured (recall) 4get/KN2 mice were infected with H. polygyrus and CD4+ T cells in the LP were analyzed for the expression of GFP and huCD2 at the indicated days.

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